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WO2013016591A1 - Isoprène synthases à efficacité élevée produites par l'ingénierie protéique - Google Patents

Isoprène synthases à efficacité élevée produites par l'ingénierie protéique Download PDF

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Publication number
WO2013016591A1
WO2013016591A1 PCT/US2012/048422 US2012048422W WO2013016591A1 WO 2013016591 A1 WO2013016591 A1 WO 2013016591A1 US 2012048422 W US2012048422 W US 2012048422W WO 2013016591 A1 WO2013016591 A1 WO 2013016591A1
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Prior art keywords
isoprene
synthase
nucleic acid
variant
isoprene synthase
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PCT/US2012/048422
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English (en)
Inventor
Thomas D. SHARKEY
Simon E. Aspland
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Zuvachem, Inc.
Board Of Trustees Of Michigan State University
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Publication of WO2013016591A1 publication Critical patent/WO2013016591A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/007—Preparation of hydrocarbons or halogenated hydrocarbons containing one or more isoprene units, i.e. terpenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/02—Preparation of hydrocarbons or halogenated hydrocarbons acyclic
    • C12P5/026—Unsaturated compounds, i.e. alkenes, alkynes or allenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/03—Carbon-oxygen lyases (4.2) acting on phosphates (4.2.3)
    • C12Y402/03027—Isoprene synthase (4.2.3.27)

Definitions

  • the invention relates, in pari, to high efficiency enzymes needed for the economic viability of enzyme driven processes for the production of renewable isoprene,
  • Isoprene is a versatile feedstock utilized in production of synthetic rubber (poly-isoprene), elastomers, lubricants, and specialty chemicals.
  • isoprene has a boiling point of 34°C, a property that offers a number of production and purification advantages.
  • Isoprene is currently produced by the petrochemical industry as a by-product of the thermal cracking of crude oil, The yield of isoprene from this process is small and requires the collection of 5-carbon molecule (C5) streams from several refineries and the separation of individual products from these streams, including isoprene. Approximately 800,000 metric tons of isoprene is produced by this route every year. Efficient polymerization of this product into poly-isoprene is handicapped by impurities that are difficult to separate from the C5 stream of thermally cracked oil.
  • ROS reactive oxygen species
  • the enzyme synthesizing isoprene preferably produces isoprene at a high rate, e.g., in a fermentative setting greater than 2,5g of isoprene per litre per hour of microbial culture, and, even more preferably, will be able to produce many times its own weight in isoprene.
  • a high rate e.g., in a fermentative setting greater than 2,5g of isoprene per litre per hour of microbial culture, and, even more preferably, will be able to produce many times its own weight in isoprene.
  • isoprene synthases In addition to their high K m , known isoprene synthases have a very low turnover constant, k cat> or overall catalytic rate. Furthermore, plant isoprene synthases did not evolve to function in an extracellular or non-plant cellular environment such as that found inside microbes grown in a fermenter. Consequently, their performance in such settings will be sub-optimal. Therefore, what is needed to meet the increasing worldwide demand for isoprene and products derived from isoprene is an isoprene synthase with optimized K m and K cai values, which efficiently synthesizes isoprene in a fermentative mileau. Further, methods for engineering one or a combination of . m and k cai values of an isoprene synthase would represent a significant advancement o ver conventional methods of the art,
  • This invention provides isoprene synthases having properties necessary' for efficient fermentation of feedstocks to produce isoprene, and protein engineering approaches for producing enzymes with an enhanced ability, relative to wild type isoprene synthases, to catalyse the formation of isoprene.
  • methods include modifying existing isoprene producing enzymes or converting related enzymes that do not natural ly produce isoprene into enzymes capable of catalyzing isoprene formation.
  • such enzymes are sufficiently efficient in the catalysis of isoprene formation to be useful in an industrial setting.
  • the present invention provides isoprene synthases able to catalyze the synthesis of isoprene with an activity greater than that found in natually occurring isoprene synthases.
  • factors contributing to the present invention are novel insights into the structure and activity of known isoprene synthases, and other closely related hemiterpenoid and terpenoid synthases.
  • the superior isoprene synthases of the in vention are produced by one or more of the following routes: (a) identification and, preferably, isolation, of a novel native isoprene synthase, (b) conversion of a methyl butenol synthase into an isoprene synthase by the addition, removal or substitution of one or more amino acids, (c) replacement of one or more unstructured arm of a characterized isoprene synthase with the arm of a snapdragon monoterpene synthase, e.g., one that makes the acyclic monoterpenes ocimene and myrcene, (d) conversion of an acyclic monoterpene synthase that removes diphosphate from an aliylic isoprenoid precursor into an isoprene synthase by substitution of one or amino acids in their active sites, e.g., with phenylalanine, tyrosine or try
  • one or more of the approaches listed abo ve are combined to produce a single novel isoprene synthase to further increase its efficiency and/or activity relative to a wild type isoprene synthase in the synthesis of isoprene.
  • the active site of the novel isoprene synthases may be modified by opening up their active sites through replacement of the phenylalanines smaller amino acids such as leucine.
  • thermostability of the novel isoprene synthase will be increased relative to the corresponding wild type.
  • the native isoprene synthases have evolved to function in terrestrial plants at temperatures that vary from the culture conditions ideal for microbes in an industrial bioreactor or fermenter.
  • the present invention provides novel isoprene synthases having optimal activity at the optimal temperature for microbes producing isoprene in a bioreactor or fermenter. f 0013] In other embodiments of the invention, it is provided variant isoprene synthases that are more resistant than the corresponding wild type enzymes to oxidative stress.
  • the novel isoprene synthases will be modified relative to the corresponding wild type enzyme to facilitate greater activity in their new setting in either microbial hosts or combinations of isolated enzymes.
  • These modifications include changing codon usage from that preferred by terrestrial plants to that preferred by the chosen microbial host.
  • These modifications optionally further include removal of cell localization sequences that direct isoprene to compartments within a terrestrial plant cell, for example, the chloroplast that microbes do not have.
  • These modifications optionally further include changes to increase the stability and solubility of the enzymes producing isoprene.
  • an isolated nucleic acid sequence having a sequence encoding an isoprene synthase variant of a parent eucalyptus isoprene synthase, said sequence operably linked to a promoter, wherein the isoprene synthase variant is capable of catalyzing a reaction that synthesizes isoprene from DM ADP fdimethyiallyl disphosphate) and said isoprene synthase variant is truncated at the N-termimis as compared to the parent eucalyptus isoprene synthase,
  • the parent eucalyptus isoprene synthase is E.
  • the isoprene synthase variant may have a sequence that has at least five, at least ten, at least fifteen, at least twenty, at least twenty five or at least thirty amino acids truncated from the N-terminus as compared to the parent eucalyptus isoprene synthase.
  • the isoprene synthase variant has a sequence that has five to thirty five, ten to thirty five, fifteen to thirty five, twenty to thirty five, twenty five to thirty five, or thirty to thirty five amino acids truncated from the N-terminus as compared to the parent eucalyptus isoprene synthase.
  • the isoprene synthase variant has a sequence that has thirty five amino acids tnudied from the N-terminus as compared to the parent eucalyptus isoprene synthase.
  • the isoprene synthase variant has a sequence that has less than thirty five amino acids truncated from the N-terminus as compared to the parent eucalyptus isoprene synthase.
  • an isolated nucleic acid sequence having a sequence encoding a variant of a parent solanum pheilandrene synthase, said sequence operably linked to a promoter, wherein the variant is capable of catalyzing a reaction that synthesizes isoprene from DMADP (dimethylaliyl)
  • the parent solanum pheilandrene synthase is S. lycopersicum of SEQ ID NO: 23.
  • the isoprene synthase variant may have a sequence that has at least five, at least ten, at least fifteen, at least twenty, at least twenty five, at least thirty, or at least thirty five amino acids truncated from the N- terniinus as compared to the parent solanum pheilandrene synthas.
  • the variant has a sequence that has five to thirty six, ten to thirty- six, fifteen to thirty six, twenty to thirty six, twenty five to thirty six, or thirty to thirty six amino acids truncated from the N-terminus as compared to the parent solanum pheilandrene synthas. In some embodiments, the variant has a sequence that has thirty six amino acids truncated from the N-terminus as compared to the parent solanum phel!andrene synthas. In still other embodiments, the variant has a sequence that has less than thirty six amino acids truncated from the N-terminus as compared to the parent solanum phellandrene synthas.
  • the promoter is, in some embodiments, a prokaryotic promoter, e.g. pTrc promoter. Those of skill in the art would be able to select other suitable prokaryotic promoter for use in the present invention. In preferred embodiments, the promoter is not a strong promoter.
  • an expression vector ocomprising the nucleic acid sequence described abo ve.
  • an isolated host cell comprising the heterologous nucleic acid sequence or expression vector described above.
  • the host cell may in some embodiments be a bacterial cell, e.g. Escherichia coli.
  • the host cell may further comprise one or more recombinant nucleic acid sequence of a MEP pathway gene, such as one or more selected from dxs, ispD, ispF, and idi.
  • an isolated isoprene synthase variant of a parent eucalyptus isoprene synthase wherein said variant comprises a truncation in the N-terminal portion of isoprene synthase as compared to the parent eucalyptus isoprene synthase and wherein said variant is capable of catalyzing a reaction that synthesizes isoprene from DMADP (dimethylailyl disphosphate).
  • a variant of a parent solanum phellandrene synthase wherein said variant comprises a truncation in the N-terminal portion of isoprene synthase as compared to the parent eucalyptus isoprene synthase and wherein said variant is capable of catalyzing a reaction that synthesizes isoprene from DMADP (dimethylailyl disphosphate).
  • methods of producing isoprene comprising: ( a) providing a host cell comprising an expression vector including the nucleic acid sequence described above; and (b) culturing the host cell under conditions suitable for producing isoprene, e.g. optionally further comprising (c) recovering the isoprene, e.g. still optionally further comprising (d) polymerizing the isoprene.
  • BRI EF DESCRIPTION OF DRAWINGS BRI EF DESCRIPTION OF DRAWINGS
  • FIGURE 1 Kinetic behavior of isoprene synthase proteins from poplar
  • FIGURE 2 Mechanism of hemiterpene synthesis from dimethylallyl diphosphate (DMADP).
  • FIGURE 3 Reaction mechanisms leading to hemiterpenes and monoterpenes. Cyclic monoterpenes require a rotation around die 2-3 bond, which is facilitated by reattaching the diphosphate to carbon 3 of the linalyS cation. This step is not required for making isoprene or acyclic monoterpenes.
  • FIGURE 4 Isoprene is made from dimethylallyl diphosphate by elimination of the diphosphate to yield a carbocation intermediate. Abstraction of any one of six protons of the two methyl groups leads to the formation of isoprene. Similar chemistry beginning with geranyl diphosphate leads to acyclic monoterpenes. Proton abstraction of any of the methyl hydrogens leads to beta myrcene while either E (trans) or Z (cis) beta ocimene is made depending upon which proton of carbon 4 is abstracted.
  • FIGURE S Structure of bornyl diphosphate synthase (PDB 1N1 Z).
  • Gray ribbons are the ⁇ -subunit helices with no direct role in catalysis.
  • the orange ribbon is helix A. of the a subunit.
  • the short sand colored stretch is the C-terminus.
  • the unstructured blac N-terminal amino acids, the orange A-helix amino acids, and the C-terminal amino acids must fold together in the region that joins the two subunits. This may be the cause of frequent misfolding found in isoprene synthases expressed in bacteria.
  • FIGURE 6 N and C termini of horny] diphosphate synthase and modeled ocimene synthase of snapdragon.
  • Black N terminus of BPPS
  • Gray modeled N terminus of ocimene synthase.
  • Purple identically located leucine residues that will serve as the cut over location for a chimeric enzyme.
  • Green is BPPS structure while blue is ocimene synthase modeled structure.
  • the sand-colored line is the C- terminus of 13PPS that does not occur in ocimene synthase and will be cleaved in the chimeric enzyme.
  • FIGURE 7 View of the binding pocket taken from 3I4X (PDB crystal structure, drawn in MacPy ol).
  • the purple color is surface provided by tyrosines
  • green are carbons of the DMADP analog DMASP.
  • Exemplary mutations to the active site include, either alone or in combination, converting the lower two tyrosines to phenylalanine and converting one or more amino acids on the facing wall to
  • FIGURE 8 The in vivo isoprene production by E. coli BL21 strains expressing either E. globulus or P. alba isoprene synthases, with or without heterologous expression of dxs, ispD, ispF, and idi, was measured in a FIS as described in Example
  • FIGURE 9 Specific activity was obtained at various DMADP concentrations for E. globulus isoprene synthase and indicated by the light grey diamonds. Data were fitted to Equation 1 using the constants shown in Table 1 and the fitted equation is shown by the dark grey line as described in Example 3.5.
  • FIGURE 10 A comparison of the relative expression and solubility of E. globulus, M. alterniforia, and R. pseudoacacia isoprene synthases is shown. Total, soluble, and insoluble protein fractions are shown after induction of the indicated plasmids described in Example 4.4 and the E. globulus isoprene synthase expressed at high levels in soluble form.
  • FIGURE 11 Specific activity was obtained according to Example 5.2 at various DMADP concentrations for S. ivcopersicum pheliandrene synthase and indicated by the plotted line.
  • “Sustainable energy,” as used herein, refers broadly to energy other than fossil fuels.
  • Exemplary sources of sustainable energy include, but are not limited to, solar energy, water power, wind power, geothermal energy, wave energy, and energy produced from other sources, such as wastes and renewables.
  • hydrocarbon compounds includes hydrocarbons and hydrocarbon derivatives, e.g., alcohol, halide, thiol, ether, aldehyde, ketone, carboxylic acid, ester, amine, and amide, etc.
  • the term "carbonaceous chemical,” refers to any carbon- containing chemical that can be produced by a biocatalyst.
  • the carbonaceous chemical is a hydrocarbon, while in other embodimenis, the chemical includes one or more heteroatoms, e.g., O, S, N, P and the like.
  • the heteroatoms can be joined to one or more carbon atoms or, when there is more than one heteroatom. they are optionally joined to each other, e.g., S0 3 H.
  • the carbonaceous chemical can include residues that are alkyl, heteroalkyl, aryl or heteroaryi residues.
  • the method and system of the invention is of use to produce a carbonaceous chemical in an "essentially pure state,"
  • the term "essentially pure state” refers to a purity of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% at least 99,5%, at least 99.9% or at least 99.95%.
  • alkyl by itself or as part of substituent, means, unless otherwise stated, a straight or branched chain, or cyclic hydrocarbon radical, or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include mono-, di- and multivalent radicals, having the number of carbon atoms designated (i.e. Ci-Cio means one to ten carbons).
  • the term “alkyl” means a straight or branched chain, or combinations thereof, which may be fully saturated, mono- or polyunsaturated and can include di- and multivalent radicals.
  • saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n- propyi, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cycloliexvl, (cyciohexyl)methyl, cyc!opropyimethyS, homologs and isomers of, for example, n-penty!, n-hexyl, n-heptyl, n-octyl, and the like,
  • An unsaturated alkyl group is one having one or more double bonds or triple bonds.
  • alkyl groups examples include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(l,4- pentadienyl), ethynyl, 1 ⁇ and 3-propynyl, 3-butynyl, and the higher homologs and isomers.
  • alkyl unless otherwise noted, is also meant to include those derivatives of alkyl defined in more detail below, such as “heteroalkyl” with the difference that the heteroalkyl group, in order to qualify as an alkyl group, is linked to the remainder of the molecule through a carbon atom.
  • Alkyl groups that are limited to hydrocarbon groups are termed "homoalkyl".
  • alkenyl by itself or as part of another substituent is used in its conventional sense, and refers to a radical derived from an alkene, as exemplified, but not limited, by substituted or unsubstituted vinyl and substituted or unsubstituted propenyl.
  • an alkenyl group will have from 1 to 24 carbon atoms, with those groups having from 1 to 10 carbon atoms being useful examplars.
  • alkylene by itself or as part of another substituent means a divalent radical derived from an alkaiie, as exemplified, but not limited, by -CH 2 CH 2 CH 2 CH 2 -, and further includes those groups described below as “heteroalkylene.”
  • an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being useful exemplars in the present invention.
  • a “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.
  • alkoxy alkyl amino
  • alkylthio or thioalkoxy
  • heteroalkyl by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or cyclic hydrocarbon radical, or combinations thereof, consisting of the stated n umber of carbon atoms and at least one heteroatom selected from the group consisting of O, N, Si, S, B and P and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. in some embodiments, the term
  • heteroalkyl by itself or in combination with another term, means a stable straight or branched chain, or combinations thereof, consisting of the stated number of carbon atoms and at least one heteroatom.
  • the heteroatom(s) may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, -CH 2 -CH 2 -O-CH 3 , -CH 2 -CH 2 -NH-CH 3 , -CH 2 -CH 2 -N(CH 3 )-CH 3 , -CH 2 -S-CH 2 -CH 3 , -CI I K-S ⁇ 0 ⁇ 1 :.
  • heteroalkylene by itself or as part of another substituent means a divalent radical derived from heteroalkyl, as exemplified, but not limited by, -CH 2 -CH 2 -S-CH 2 -CH 2 - and ⁇ H 2 -S-CH 2 -CH 2 -NH-CH 2 - ,
  • heteroatoms can also occupy either or both of the chain termmi (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like).
  • no orientation of the linking group is implied by the direction in which the formula of the linking group is written.
  • the formula -C0 2 R'- represents both -C(0)OR' and -OC(0)R'.
  • a heteroatom can occupy the position at which the heterocycie is attached to the remainder of the molecule
  • a "cycloalkyl” or “heterocycloalkyl” substituent may be attached to the remainder of the molecule directly or through a linker, wherein the linker is preferably a!ky!ene.
  • eyeloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like.
  • heterocycloalkyl examples include, but are not limited to, l -(l,2,5,6-tetrahydropyridyl), 1- piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran- 2 ⁇ yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2- piperazinyl, and the like.
  • halo or halogen, by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.
  • haloalkyl are meant to include monohaloalkyl and polyhaloaikyi.
  • halo(C 1 -C 4 )a3kyl is mean to include, but not be limited to, trifluoromethyi, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.
  • aryl means, unless otherwise stated, a polyunsaturated, aromatic, substituent that can be a single ring or multiple rings (preferably from 1 to 3 rings), which are fused together or linked covalently.
  • heteroaryl refers to aryl groups (or rings) that contain from one to four heteroatoms selected from N, O, S, Si and B, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized.
  • a heteroaryl group can be attached to the remainder of the molecule through a heteroatom.
  • Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3- pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-irnidazoryl, pyrazinyl, 2-oxazolyl, 4-oxazo y , 2- phenyl-4-oxazolyi, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4- thiazolyi, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyi, 3-pyridyl, 4- pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5- indoly
  • aryl when used in combination with other terms (e.g., aryloxy, arylthioxy, arylalkyl) optionally includes both aryl and heteroaryl rings as defined above.
  • arylalkyl is meant to include those radicals in which an aryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl and the like) including those alky!
  • a carbon atom e.g., a methylene group
  • an oxygen atom e.g., pheiioxymethyl, 2- pyridyloxymethyS, 3-(l-naphthyloxy)propyl, and the like.
  • alkyl e.g., "alkyl,” “heteroalkyl,” “a yl” and “heteroaryl” are meant to include both substituted and unsubstituted forms of the indic ated radical.
  • exemplary substituents for each type of radical are provided belo w.
  • R', R", R'" and R" each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, e.g., aryl substituted with 1-3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups.
  • each of the R groups is independently selected as are each R', R", R'" and R"" groups when more than one of these groups is present.
  • R' and R" When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.
  • -NR'R is meant to include, but not be limited to, 1-pyrrolidinyl and 4- morpholiny!.
  • alkyl is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g., -CF 3 and -CH 2 CF 3 ) and acyl (e.g., -C(0)CH 3 , -C(0)CF 3 , -C(0)CH 2 OCH 3 , and the like).
  • substituents for the aryl and heteroaryl groups are genetically referred to as "aiyl group substituents.”
  • each of the R groups is independently selected as are each R', R", R' " and R"" groups when more than one of these groups is present.
  • R groups are independently selected as are each R', R", R' " and R"" groups when more than one of these groups is present.
  • Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -T-C(0)-(CRR') q -U-, wherein T and U are independently -NR-, -0-, -CRR' ⁇ or a single bond, and q is an integer of from 0 to 3.
  • two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH 2 ) r -B-, wherein A and B are independently --CRR'-, -0-, - R-, -S-, -S(0)-, ⁇ S(0) 2 ⁇ , -S(0) 2 ' NR'- or a single bond, and r is an integer of from 1 to 4.
  • One of the single bonds of the new ring so formed may optionally be replaced with a double bond.
  • two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -(CRR') s -X-(CR"R'") c i-, where s and d are
  • R, R', R" and R'" are preferably independently selected from hydrogen or substituted or unsubstituted (CrC 6 )alkyl.
  • acyl describes a substituent containing a carbonyl residue, C(Q)R.
  • R exemplary species for R include H, halogen, substituted or unsubstituted a!kyl, substituted or unsubstituted ary!, substituted or unsubstituted heteroaryl, and substituted or unsubstituted iieterocycloaikyi,
  • fused ring system means at least two rings, wherein each ring has at least 2 atoms in common with another ring.
  • “Fused ring systems” may include aromatic as well as non-aromatic rings. Examples of “fused ring systems” are naphthalenes, indoles, quinoiines, chromenes and the like.
  • heteroatom includes oxygen (O), nitrogen (N), sulfur (S), silicon (Si) and boron (B).
  • R is a general abbreviation that represents a substituent group.
  • substituent groups include substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted iieterocycloaikyi groups.
  • amino acid and “amino acid identity” as used herein is meant one of the 20 naturally occurring amino acids or any non-natural analogues that may be present at a specific, defined position.
  • protein herein is meant at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides.
  • the protein may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures, i.e. "analogs”, such as peptoids (see, Simon et al., PNAS USA 89(20):9367 (1992)) particularly when LC peptides are to be administered to a patient.
  • amino acid or “peptide residue”, as used herein means both naturally occurring and synthetic amino acids. For example, homophenylalanine, citrulline and noreleucine are considered amino acids for the purposes of the invention.
  • Amino acid also includes imino acid residues such as proline and hydroxyproline.
  • the side chain may be in either the (R) or the (S) configuration, in the preferred embodiment, the amino acids are in the (S) or L-configuration. If non- naturally occurring side chains are used, non-amino acid substituents may be used, for example to prevent or retard in vivo degradation.
  • starting gene and “parent gene” refer to a nucleic acid which is a gene of interest that encodes a protein of interest that is to be improved and/or changed using the present invention.
  • starting protein and “parent protein” refer to a protein of interest that is to be improved and/or changed using the present invention.
  • the nucleic acid is a recombinant nucleic acid.
  • an isoprene synthase nucleic acid is operably linked to another nucleic acid encoding all or a portion of another polypeptide such that the recombinant nucleic acid encodes a fusion polypeptide that includes an isoprene synthase and all or part of another polypeptide (e.g., a peptide that facilitates purification or detection of the fusion polypeptide, such as a His-tag).
  • part or ail of a recombinant nucleic acid is chemically synthesized.
  • the nucleic acid is a heterologous nucleic acid.
  • heterologous nucleic acid is meant a nucleic acid whose nucleic acid sequence is not identical to that of another nucleic acid naturally found in the same host cell.
  • the nucleic acid includes a segment of or the entire nucleic acid sequence of any naturally-occurring isoprene synthase nucleic acid.
  • the nucleic acid includes at least or about 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, or more contiguous nucleotides from a naturally-occurring isoprene synthase nucleic acid.
  • the nucleic acid has one or more mutations compared to the sequence of a wild-type (i.e., a sequence occurring in nature) isoprene synthase nucleic acid.
  • the nucleic acid has one or more mutations (e.g., a silent mutation) that increase the transcription or translation of isoprene synthase nucleic acid.
  • the nucleic acid is a degenerate variant of any nucleic acid encoding an isoprene synthase polypeptide.
  • An isoprene synthase nucleic acid can be incorporated into a vector, such as an expression vector, using standard techniques (Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 2001 , which is hereby incorporated by reference in its entirety, particularly with respect to the screening of appropriate DNA sequences and the construction of vectors), Methods used to ligate the DN A construct comprising a nucleic acid of interest such as isoprene synthase, a promoter, a terminator, and other sequences and to insert them into a suitable vector are well known in the art, Additionally, vectors can be constructed using known recombination techniques (e.g., Invitrogen Life Technologies, Gateway Technology), [0062] As used herein, "homologous genes” refers to a pair of genes from different, but usually related species, which correspond to each other and which are identical or very similar to each other. The term encompasses genes that are separated by speciation (i.e., the development
  • ortholog and “orthoiogous genes” refer to genes in different species that have evolved from a common ancestral gene (i.e., a homologous gene) by speciation. Typical ly, orthologs retain the same function during the course of evolution. Identification of orthologs finds use in the reliable prediction of gene function in newly sequenced genomes.
  • paralog and paralogous genes refer to genes that are related by duplication within a genome. While orthologs retain the same function through the course of evolution, paraiogs evolve new functions, even though some functions are often related to the original one.
  • homology refers to sequence similarity or identity, with identity being preferred. This homology is determined using standard techniques known in the art (See e.g., Smith and Waterman, Adv Appl Math, 2:482, 1981 ; Needleman and Wunsch, J Mol Biol, 48:443, 1970; Pearson and Lipman, Proc Natl Acad Sci USA, 85:2444, 1988; programs such as GAP, BESTFIT, FASTA, and TFASTA in the
  • an "analogous sequence" of an isoprene synthase is one wherein the function of the gene is essentially the same as the gene based on the kudzu isoprene synthase. Additionally, analogous genes include at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity with the sequence of the kudzu isoprene synthase, in additional embodiments more than one of the above properties applies to the sequence. Analogous sequences are determined by known methods of sequence alignment. A commonly used alignment method is BLAST, although as indicated above and below, there are other methods that also find use in aligning sequences.
  • Percent sequence identity refers to the percentage of residues that are identical in the two sequences when the sequences are optimally aligned.
  • 80% amino acid sequence identity means that 80% of the amino acids in two optimally aligned polypeptide sequences are identical
  • substantially identical in the context of two nucleic acids or polypeptides thus refers to a polynucleotide or polypeptide that comprising at least 70% sequence identity, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97%, preferably at least 98% and preferably at least 99% sequence identity as compared to a reference sequence using the programs or algorithms (e.g., BLAST, ALIGN, CLUSTAL) using standard parameters.
  • One indication that two polypeptides are substantially identical is that the first polypeptide is immunologically cross-reactive with the second polypeptide.
  • polypeptides that differ by conservative amino acid substitutions are immunologically cross-reactive.
  • a polypeptide is substantially identical to a second polypeptide, for example, where the two peptides differ only by a conservative substitution.
  • Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions (e.g., within a range of medium to high stringency).
  • polypeptides includes polypeptides, protems, peptides, fragments of polypeptides, and fusion polypeptides that include part or all of a first polypeptide (e.g., an isoprene synthase) and part or all of a second polypeptide (e.g., a peptide that facilitates purification or detection of the fusion polypeptide, such as a His-tag).
  • a polypeptide has at least or about 50, 100, 150, 175, 200, 250, 300, 350, 400, or more amino acids.
  • the polypeptide fragment contains at least or about 25, 50, 75, 100, 150, 200, 300, or more contiguous amino acids from a full-length polypeptide and has at least or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,, 90%, 95%, 100% or greater than 100% of an activity of a corresponding full-length polypeptide.
  • the polypeptide includes a segment of or the entire amino acid sequence of any naturally-occurring isoprene synthase.
  • the polypeptide has one or more mutations compared to the sequence of a wild-type (i.e., a sequence occurring in nature) isoprene synthase.
  • the polypeptide is a heterologous polypeptide.
  • heterologous polypeptide is meant a polypeptide whose amino acid sequence is not identical to that of another polypeptide naturally expressed in the same host ceil.
  • variant polypeptide as used herein is meant a polypeptide sequence that differs from that of a parent polypeptide sequence by addition, deletion or substitution of at least one amino acid modification.
  • Variant polypeptide may refer to the polypeptide itself, a composition comprising the polypeptide, or the nucleic acid sequence that encodes it.
  • the variant polypeptide has at least one amino acid modification compared to the parent polypeptide, e.g. from about one to about ten amino acid modifications, and preferably from about one to about five amino acid modifications compared to the parent.
  • variant polypeptide sequence herein will preferably possess at least about 80% identity with a parent polypeptide sequence, and most preferably at least about 90% identity, more preferably at least about 95% identity, Accordingly, by "isoprene synthase variant” as used herein is meant an isoprene synthase sequence that differs from that of a parent isoprene synthase sequence by virtue of at least one amino acid modification.
  • an active site refers to a region of a polypeptide or a molecular complex comprising the polypeptide that, as a result of the primary amino acid sequence of the polypeptide and/or its three-dimensional shape, favorably associates with another chemical entity or compound including ligands or inhibitors.
  • an active site may include or consist of features such as interfaces between domains.
  • Chemical entities or compounds that may associate with an active site include, but are not limited to, compounds, ligands, cefaclors, substrates, inhibitors, agonists, antagonists, etc.
  • Structural reaction residues and "site-construction residues” refer to a three- dimensional collection of amino acids involved in an enzymatic reaction. For example, these would include those forming the active site, those coordinating metal ions and those forming the substrate bind region. In particular, those forming the flexible loops and N-terminus and the adjacent residues that stabilize the flexible segments when substrate is bound, in an exemplar ⁇ ' embodiment, the invention provides an enzyme with isoprene synthase activity that is a variant polypeptide modified by addition, deletion or substitution of at least one amino acid that is a structural reaction residue.
  • equivalent or homologous residues refers to amino acid residues that are shared by certain proteins. Equivalent residues may be identified by determining homology at the level of tertiary stnicture for a terpene synthase (e.g., isoprene synthase) whose tertiary structure has been determined by x-ray
  • Equivalent residues are defined as those for which the atomic coordinates of two (2) or more of the main chain atoms of a particular amino acid residue of the terpene synthase having putative equivalent residues and the substrate of interest (e.g., N on N, CA on CA, C on C and O on O) are within 0.2 nm and preferably 0.15 nm after alignment. Alignment is achieved after the best model has been oriented and positioned to give the maximum overlap of atomic coordinates of non-hydrogen protein a toms of the terpene synthases and subs trates analyzed.
  • the preferred model is the crystallographic model giving the lowest R factor for experimental diffraction data at the highest resolution available, determined using methods known to those skilled in the art of crystallography and protein characterization/analysis.
  • equivalent residues which are functionally analogous to a specific residue of isoprene synthase are defined as those amino acids at a structurally homologous synthase which may adopt a conformation such that they either alter, modify, or contribute to protein stnicture, substrate binding or catalysis in a manner defined or attributed to a specific residue of isoprene synthase.
  • Biocatalyst refers to a microbe that is converts a carbon source into a product, in this case isoprene, that is expelled from the microbe that produced it.
  • Embodiments of the present in vention provide a key component of ne w energy and chemical industry solutions that are not dependent on fossil fuels and are
  • the present invention provides compositions for producing isoprene.
  • the composition includes no vel enzymes capable of producing isoprene, some of which are isoprene synthases, that have favorable characteristics in isolation or when expressed in a biocataiyst. In either case they are useful for the efficient and economical production of isoprene from a variety of carbonaceous feedstocks.
  • Isoprene synthase genes have been cloned and characterized from two plant genera: Populus (poplars and aspen) (Miller, et al., (2001) Planta 213: 483-487; Sharkey, et al, (2005) Plant Physiology 137: 700-712; Sasaki, et al, (2005) FEES Letters 579: 2514-2518 and Behnke, et al., (2007) The Plant Journal 51 : 485-499) and Pueraria montana (kudzu) (Sharkey, et al., (2005) Plant Physiology 137: 700-712).
  • the enzyme catalyzes the divalent cation-dependent, irreversible, and stoichiometric conversion of dimethyiallyi diphosphate (DMADP) to isoprene and pyrophosphate.
  • DMADP dimethyiallyi diphosphate
  • the wild type protein first purified from aspen leaves, was determined to be functional as a heterodimer (Sil ver and Fall, (1995 ) J. Biological Chemistry 270: 13010-13016).
  • the associated monomers were found to be similar, but not identical (62 and 58 kDa). However, monomers and/or homodimers have activity since single gene products can make isoprene.
  • the native enzyme is sensitive to denaturation due to dilution as well as oxidation, and therefore the samples were kept concentrated and in a high concentration of DTT. This suggests that a free thiol may be required for catalytic activity.
  • the enzyme required a divalent cation for activity, either Mg r or Mn' + .
  • Kinetic analysis of the native enzyme demonstrates maximal activity at pH 8.0.
  • the apparent k cat and K m for the enzyme were estimated to be 1.7 s "1 and 8 niM respectively, but the enzyme also exhibited significant substrate inhibition (Eq. 1).
  • the highest activity of the native protein was estimated to be 900 nmol isoprene min 1 mg-1 a t 10 raM DMADP.
  • Equation 1 Enzymatic rate equation for enzyme with substrate inhibition.
  • the hybrid poplar protein and its recombinant versions were all monomelic, although the purification conditions were different from the previous work with the aspen enzyme. This suggests that the heterodimer observed in the aspen leaves may be comprised of alternatively modified versions of the same gene product.
  • the addition of the polyhistidine tags to the protein significantly affected the activity of the recombinant isoprene synthase protein. All of the proteins exhibited a broad pH optimum of 8.0 except for the protein with N-terminal tag, which exhibited a pH optimum of 9.0. All of the enzymes had temperature optima of 40 °C except for the protein with the ( "' -terminal tag, which was more thermostable (optimal activity at 45-50 °C).
  • Equation 3 a modified Michaelis-Menten equation allowing for both
  • the recombinant enzyme from Populus alba was not expressed with a polyhistidine tag, and was found to be very similar to that of the hybrid poplar (Sasaki, et al, (2005) FEBS Letters 579: 2514-2518). This protein was also monomelic, had a pH optima of 8.0, a temperature optima of 40 °C, and an apparent . m of 8.7 mM. No kc at was determined.
  • the RR is required for the first steps of the reaction.
  • the diphosphate is released from the end of GDP making a linalyi cation ( Figure 3).
  • the diphosphate is reattached at carbon 3, This reattachment ensures that the bond between carbons 2 and 3 is a single bond, and this allows rotation around that bond required to convert the linalyi cation to a neryl cation.
  • the neryl cation is the immediate precursor of cyclic monoterpenes (e.g. limonene). Proton abstraction leads to quenching of the cation and typically bond formation between carbons 1 and 6 making a six-membered ring.
  • acyclic monoterpenes can be formed from the linalyi cation.
  • trichomes of tomato have a enzyme (a phellandrene synthase) that uses neryl diphosphate (NDP), the cis isomer of GDP (Schilmiller et al, (2009) PNAS 106: 10865-10870).
  • NDP neryl diphosphate
  • This enzyme forms the neryl cation as soon as the diphosphate is removed and so does not require the rotation around the 2-3 bond required when G DP is the starting material. This simplifies the reaction mechanism.
  • This enzyme can use GDP at a low rate.
  • phellandrene synthase makes various products.
  • NDP is the substrate only cyclic products are made but when GDP is the substrate some acyclic monoterpenes are made. This indicates that the acyclic monoterpenes are made from the linalyi cation and when NDP is supplied the neryl cation is not converted to the linalyi cation.
  • Physcomatrella patens has a similar mechanism in which a cation is quenched by proton abstraction. Most kaurene synthases make only kaurene but the P. patens enzyme will sometimes quench the cation with water resulting in kaurane (Kawaide et al, (2011) FEBS Journal. 278: 123-133). A similar reaction in a hemiterpene synthase would produce methyl butenol. Unpublished sequences of methyl butenol synthase show a similar amino acid substitution as the P, patens kaurane synthase and may explain why this enzyme makes methyl butenol instead of isoprene.
  • phenylalanines line the active site. This makes the active site rich in pi electrons (from the double bonds in phenylalanine).
  • a strong but often overlooked molecular interaction is cation-pi interactions (Dougherty, (1996) Science 271: 163-169; Gallivan and
  • the present invention provides an isoprene synthase in which at least one of these parameters are improved.
  • one or both of these parameters is impro ved by a t least about 1, by at least about two or by at least about three orders of magnitude.
  • the improved k cai provides higher yields, e.g., at maximum substrate availability.
  • the lower m provides advantages in the synthesis of isoprene, e.g., the alleviation of probable feedback on substrate production.
  • the improved K m enhances the ease of steering carbon into isoprene production.
  • the invention provides enzymes with isoprene synthase activity with modified stability and/or solubility (e.g., in the fermentation medium or intracellular mileau), thereby providing an increased le vel of isoprene synthase that can be expressed in a cell or in vitro system.
  • the alterations to stability and/or solubility increase isoprene synthase activity in a cell or in vitro systems by at least about an order of magnitude, at least about two orders of magnitude or at least about three orders of magnitude. It is likely that a total increase in isoprene synthase activity in a cell or in vitro system of approximately three orders of magnitude will be necessary to make bio-catalyst driven production of isoprene economically viable.
  • the present invention provides enzymes with an isoprene synthase activity that is at least about one order of magnitude greater, at least about two orders of magnitude greater and at least about three orders of magnitude greater in a cell or in vitro environment than the corresponding activity in the parent polypeptide or provide a platform for directed evolution or structure-based prediction of additional changes that will achieve such favourable kinetics.
  • the invention provides a novel polypeptide with isoprene synthase activity.
  • the invention provides a variant polypeptide with isoprene synthase activity based on a parent methyl butenol synthase polypeptide or other hemiterpene synthase from gymnosperm species converted by addition, deletion or substitution of one or more amino acid into a polypeptide with isoprene synthase activity.
  • the invention provides a variant polypeptide with isoprene synthase activity.
  • the unstructured arm of isoprene synthase is replaced with the structured arm of myrcene synthase in a variant
  • Tps-g group of monoterpene synthases (Dudareva et al., (2003) The Plant Cell 15: 1227-1241) are the seventh terpenoid synthase subfamily identified. The subfamilies identified earlier are designated Tps-a through 7 ? -/(Bohimami et al., (1998) PNAS 95: 4126-4133).
  • the Tps-g subfamily makes acyclic monoterpenes, presumably because they have a ver different initial domain, in that they lack the RRxgW motif, which is a characteristic feature of the other families of monoterpene synthases: the Tsp- b family of angiosperm monoterpene synthases and the Tsp-d family of conifer monoterpene synthases.
  • This initial domain closes off the active site and likely moves during catalysis. It is often unresolved in crystal structures but is resolved in PDB ⁇ 1 ⁇ ( Figure 5).
  • Tps-g enzymes Lack of the RR in Tps-g enzymes likely prevents the linalyl to neryl cation conversion required for cyclic monoterpenes but this conversion is not required for acyclic synthases or hemiterpene synthases.
  • This arm is likely to be a significant component of the difficulty in expressing isoprene synthases because of its flexibility and lack of secondary structure.
  • Chimeric proteins based on the native isoprene synthases are generated with the long initial arm replaced by amino acids found in Tps-g genes. These genes have a very different initial sequence with amino acids that may help form secondary structure. Homology modeling is used to identify the exact location for switching from the isoprene synthase gene sequence to the Tps-g sequence.
  • fusion should occur at a conserved leucine at the base of the long arm of all isoprene synthases.
  • the arm substitution is designed to help solubility and has potential to improve the activity in other ways including but not limited to: not allowing the linalyl to neryl rotation that might slow down the enzyme.
  • the invention provides a variant polypeptide with isoprene synthase activity.
  • Enzymes according to this embodiment are produced by converting a parent acyclic monoterpene synthase polypeptide into an isoprene synthase by adding, deleting or substituting one or more amino acid residues.
  • enzymes that start with diphosphate removal from an allylic isoprenoids precursor can be converted into a variant polypeptide with isoprene synthase activity
  • Suitable families of enzymes belong to the following named groups further identified by their Enzyme Commission (EC) Numbers. Enzymes that remove diphosphate from geranyl diphosphate including: myrcene synthases (EC 4.2.3.15), S-linalool synthases (EC 4.2.3.25) and R-linalool synthases (EC 4.2.3.26).
  • Enzymes that remove diphosphate from farnesyl diphosphate including: a-farnesene synthase (EC 4.2.3.46 ⁇ and ⁇ -farnesene synthase (EC 4.2.3.47), (3S, 6E) nerolidol synthase (EC 4.2.3.48) and (3R, 6E) nerolidol synthase (EC 4.2.3.49).
  • Enzymes that remove diphosphate from neryi diphosphate including: ⁇ -phellandrene synthase (EC 4.2.3.52).
  • one or more amino acids are substituted that line the active site, at the bottom of the pocket that would accommodate DMADP, with phenylalanine, tyrosine, or tryptophan to increase the surface area of the active site that is accounted for by pi electrons.
  • Homology modeling may further refine the predictions of which amino acids should be converted into which other amino acids.
  • the invention provides an enzyme with isoprene synthase activity by pro viding a variant polypeptide derived from parent pheilandrene synthase polypeptide.
  • the phellandrene synthase is converted, by variation (addition, deletion or substitution) of one or more amino acid residues in the protein.
  • the amino acid is in the active site.
  • Many monoterpenes are produced by monoterpene synthases from the prenyl diphosphate precursor geranyl diphosphate (G PP) in which two isoprene units are joined in the trans (E) configuration.
  • PHS1 monoterpene synthase is a ⁇ -phellandrene synthase (EC 4.2.3.52) called PHS1
  • PHS1 has a high k cat producing more than 4 molecules of phellandrene per second using NPP as a substrate.
  • Phellandrene synthase is different from typical cyclic monoterpene synthases in that it does not have the RR at the beginning of the active site. The reason for this is that it does not have to do the linalyl to neryl cation chemical conversion that most cyclic monoterpene synthases have to do. Based on homology to the stracture of isoprene synthases, it is converted into an isoprene producing enzyme by either or both of the mutations V524F and M672F (SEQ ID NO: 23).
  • the invention provides a variant polypeptide in which the pi-electron structure of the active site of the relevant parent polypeptide is varied to lower m , increase k cat or both, or to convert an enzyme without (or with minimal) isoprene synthase activity into an enzyme with isoprene synthase activity.
  • IspS isoprene synthase
  • Populus alba IspS F338 and F485 SEQ ID NO: 25
  • Populus nigra IspS F338 and F485 SEQ ID NO: 26
  • Populus trichocarpa IspS F301 and F448 SEQ ID NO: 27
  • Pueraria Montana IspS F343 and F493 SEQ ID NO: 28
  • Eucalyptus globulus IspS F326 and F473 SEQ ID NO: 29
  • Melaleuca alternifolia 1326 and F473 SEQ ID NO: 30
  • Robinia pseudoaccacia IspS F281 and F428 SEQ ID NO: 31
  • the active site is examined to find other potential locations for a phenylalanine or other pi-electron contributing amino acids in ways that do not constrict the active site. Variation in the location of phenylalanines has been found in unpublished sequences of isoprene synthases from spruce F337 and F541 (Picea pungens) (SEQ ID NO: 10) and hops F354 and F530 (Humulus lupulus) (SEQ ID NO: 32) and indicating that it should be possible to vary the location of the pi-electron contributing amino acids without destroying enzyme activity.
  • the present invention provides a variant polypeptide of a parent dimethyiallyl tryptophan synthase with isoprene synthase activity.
  • Exemplary enzymes according to this embodiment have one or more amino acid remo ved, added or substituted relative to the amino acid sequence of the parent.
  • DMADP dimethylally diphosphate
  • a cation intermediate is all good targets for conversion into isoprene synthases.
  • DMADP dimethylally diphosphate
  • This protein uses DM ADP and has a high (poor) K m (8 ⁇ ) and higher k cat (0.37 per second).
  • the structure of this enzyme is known (3I4X, PDB) ( Figure 7) and it is known to use a carbocation intermediate of DMADP similar to that in the isoprene synthase reaction (Luk and Tanner, (2009) J Am Chem Soc 131: 13932-13933).
  • the binding pocket residues on dimethylallyl tryptophan synthase are L81, T82, R83, Y191, Y345 and Y398 (SEQ ID NO: 33). One, two, three, four, five or al l six of these residues are converted to phenylalanine.
  • the families of enzymes that use dimethylaliy diphosphate (DMADP) and a cation intermediate and can be converted into isoprene synthases to form a variant polypeptide include the following identified by name and enzyme commission (EC) number include but are not limited to: dimethylallyltranstransferases (EC 2.5.1.1 ), geranyltranstransf erases (EC 2.5.1.10), trans-octaprenyltranstransferases (EC 2.5.1.1 1), adenylate dimethylallytranstransferases (EC 2.5.1.27), dimethylallylcistransferases (EC 2.5,1.28), famesyltranstransferase (EC 2.5,1.29), trihydroxypterocarpan
  • dimethylallyltransferases EC 2.5.1.33
  • dimethylallytryptophan synthase EC 2,5.1 ,34
  • chyrsanthemyl diphosphate synthase EC 2.5.1.6
  • lavandulyl diphosphate synthase EC 2.5.1.11.
  • the invention provides a variant polypeptide of a parent pentalenene synthase that produces isoprene,
  • the variant is prepared by the addition, deletion or substitution of one or more amino acid relative to the parent polypeptide,
  • Pentalenene is a complex tricyclic sesquiterpene (a terpenoid containing 15 carbon atoms), that is produced from farnesyl diphosphate by the action of pentalenene synthase (EC. 4.3.2.7). Pentalenene, is the hydrocarbon precursor of the
  • Pentaleno lactone family of antibiotics The crystal structure of pentalene synthase has been resolved (Lesburg et al., (1997) Science 277: 1820-1824), revealing the fact that it is unusually a single domain terpenoid synthase. For this reason it may behave much better in bacteria and in other ways be more stable.
  • Pentalenene synthase of Streptomyces sp. UC53 I9 (SEQ ID NO: 34) is converted into an isoprene synthase by converting V179 and/or N219 into a F, W or Y (SEQ ID NOs: 35, 36 and 37).
  • Example 1 Isoprene synthase expression and purification
  • bacterial expression constructs N-terminal or C-terminal flag tagged, capable of regulated expression of recombinant proteins described in this disclosure are generated,
  • the backbone vector used is the pET vector system (Novagen) or similar,
  • the gene sequences are codon optimized for bacterial expression and synthetically generated to match the amino acid sequences in this disclosure with a Flag tag.
  • the synthetically created gene sequences is then sub-cloned using unique, engineered restriction sites into a commercially-available expression construct (e.g. Novagen) for expression of the protein.
  • a commercially-available expression construct e.g. Novagen
  • the expression constructs generated are used to transform E. coli BL21(DE3) to generate clones for expression screening. Following the selection of the best expressing clones, induction experiments (3hrs and overnight, 37°C and 18°C) are performed on the selected clones. SDS-PAGE or immunoblot is run to compare protein expression (total and soluble) and the optimal parameters for soluble expression are chosen for production at the IE scale. The selected clones are grown in LB medium and the cultures are induced at an OD between 0.8 and 1.0. After induction, cell paste is harvested by centrifugation and stored at -20°C until purification. SDS-PAGE or Western blot is performed on an analytical sample to confirm the protein expression.
  • Recombinant protein is purified from cell paste using Flag affinity
  • enzymatic reaction rates are needed at varying substrate concentrations. Since this enzyme may display substrate inhibition and/or substrate cooperativeness (Eq. 1, Eq. 2 and together Eq. 3), it will be important to collect appropriate data in order to distinguish between, and quantify the impacts of, these effects.
  • Initial rates of the enzymes are determined with varying initial DMADP concentrations. In order to ensure that only initial rates are estimated, the rates are calculated only during the consumption of the first 10% of the initial DMADP substrate. At least 4 values above and below the K m of the enzyme are used so that an accurate estimation of the kinetic parameters can be made.
  • the experimental data is fit to the appropriate enzymatic rate equations (Eq. 1, Eq. 2 and together Eq. 3) using a least squares method. If the data matches predictions that both substrate inhibition and positive cooperativeness are present, then Equation 3 will be used.
  • Hemiterpene and other terpenoid synthases may produce more than one product.
  • samples of the product of these enzymes are adsorbed onto a Solid Phase Micro Extraction (SPME) fiber and then the fiber is heated in a GC MS.
  • SPME Solid Phase Micro Extraction
  • E. globulus isoprene synthase (except the N terminal plastid targeting sequence, amino acids 1 to 35) was codon optimized for is. coli expression and synthesized by GenScript. A 1.6 kb Ncol to Xhol fragment was cloned into the Ncol and Xhol sites of pTrcHis2B to yield pTrcHis2B-E2]spS (SEQ ID NO:39).
  • E. globulus isoprene synthase (except the N terminal plastid targeting sequence, amino acids 1 to 35) with a C terminal linker and 6 histidine motif was codon optimized for E. coli expression, synthesized, and cloned into pJexpress401 by DNA2.0 (SEQ ID NO:40, pJExpress401-E4IspS-His).
  • E. coli MEP pathway genes (dxs, ispD, ispF, and idi) were placed under control of the pTrc promoter and cloned into the Smal site of pCL1920 (SEQ ID NO:41, pCL-SDFi).
  • Example 3.3 In vivo isoprene production
  • the seed culture w r as diluted to OD600 of 0.1 in 30 mL mineral medium plus 5 g/L glucose, 1 g/L yeast extract, appropriate antibiotics, and 0.4 mM IPTG and incubated at
  • E. globulus isoprene synthase when expressed in E. coli cells, produces more isoprene than the P. alba isoprene synthase ( Figure 8).
  • globulus isoprene synthase inverted triangles
  • heterologous MEP genes and P. alba isoprene synthase (circles)
  • P. alba isoprene synthase squares
  • Example 3.4 E. globulus isoprene synthase protein purification
  • Frozen pellets were resuspended in lysis buffer (50 mM NaH 2 P0 4 , 300 mM NaCl, 10 mM imidazole, pH 8.0 with Roche Mini EDTA-free tablets per the
  • Ceil lysates were treated with RNAse A and DNAse I (Qiagen) and cleared by centrifugation. Cleared lysates were incubated with nickel NT A resin for 1 hour at 4 C then loaded into a disposable chromatography column. The column was washed with 5 times the bed volume with wash buffer (50 mM Xai l -PO ;. 300 mM NaCl, 10 mM imidazole, pH 8.0) then eluted with elution buffer (50 mM NaH 2 P0 4 , 300 mM NaCl. 250 mM imidazole, pH 8.0). Glycerol was added to 15% and the samples flash frozen in liquid nitrogen and stored at -80 C.
  • wash buffer 50 mM Xai l -PO ;. 300 mM NaCl, 10 mM imidazole, pH 8.0
  • elution buffer 50 mM NaH 2 P0 4 , 300 mM NaC
  • Example 3.5 In vitro protein kinetics
  • reaction was allowed to occur in a sealed tube at 37 C for 12 minutes, after which 1 mL of headspace was injected into a F1S for measurement of the isoprene produced. Controls with no added protein were used as appropriate to subtract nonspecific isoprene production. Specific activity measurements were fitted to Equation 1 to determine KM, k ca t, and Kjs.
  • Equation 1 Enzymatic rate equation for enzyme with substrate inhibition.
  • Table 1 Kinetic parameters of purified E. globulus isoprene synthase.
  • the published K M values for various non-Eucalyptus isoprene synthases range from 0.3 mM to 9 mM (Rasulov, et al., (2009) Plant Physiology 149: 1609-1618 and references therein and Sasaki, et al, (2005) FEBS Letters 579: 2514-2 18).
  • the K M value of 0.03 mM for E. globulus favorably compares to that of other published isoprene synthases, such as those from P, alba and other Poplar species, and thus represents a superior isoprene synthase for the commercial production of isoprene.
  • the M. altemifolia isoprene synthase (SEQ ID NO 7, except the N terminal plastid targeting sequence, amino acids 1 to 32) with a C terminal linker and 6 histidine motif (ENLYFQSGSGSG SG HHHHHH) was codon optimized for E. coli expression and synthesized by DNA2.0, A BsmBI/XhoI fragment containing the isoprene synthase was then ligated into an Ncol/Xhol digest of pTrcHis2B to yield pTrc-Ml-6His. pTrc-Rl-6His
  • the R. pseudoaccacia isoprene synthase (SEQ ID NO 8) with a C terminal linker and 6 histidine motif (ENL YFQSGSGSGSGHHHHHH) was codon optimized for E. coli expression and synthesized by DNA2.0.
  • An Ncol/Xhol fragment containing the isoprene synthase was then ligated into an Nco!/Xhol digest of pTrcHis2B to yield pTrc- R 1 -61 lis.
  • A. majus myrcene synthase (SEQ ID NO 13, except the N terminal plastid targeting sequence, amino acids 1 to 45) with a C terminal linker and 6 histidine motif (ENLYFQSGSGSGSGHHHHHH) was codon optimized for E. coli expression, synthesized, and cloned into pJexpress404 by DNA2.0. Variants were made with the indicated amino acid substitutions. Dimeihylallyl tryptophan synthase
  • A. f migatus dimeihylallyl tryptophan synthase (SEQ ID NO 33) with a C terminal linker and 6 histidme motif (ENLYFQSGSGSGSGHHHHHH) was codon optimized for E. coli expression, synthesized, and cloned into pJexpress404 by DNA2.0. Variants were made with the indicated amino acid substitutions, with "all” representing L81 F, T82F, R83F, Y191F, Y345F, and Y398F.
  • S. lycopersicum phellandrene synthase (SEQ ID NO 23, except the N terminal plastid targetmg sequence, amino acids 1 to 36) with a C terminal linker and 6 histidme motif (ENLYFQSGSGSGSGHHHHHH) was codon optimized for E. coli expression, synthesized, and cloned into pJexpress4Q4 by DNA2.0. Variants were made with the indicated amino acid substitutions,
  • the P. sabiniana methyl butanol synthase (SEQ ID NO 9, except the N terminal plastid targeting sequence, amino acids 1 to 43) with a C terminal linker and 6 histidine motif (ENLYFQSGSGSGSGHHHHHH) was codon optimized for E. coli expression, synthesized, and cloned into pJexpress404 by DNA2.0. Variants were made with the indicated amino acid substitutions,
  • Strepiomyces sp. UC5319 pentalenene synthase (SEQ ID NO 34) with a C terminal linker and 6 histidine motif ( ENLYFQSGSGSGSGHHHHHH) was codon optimized for E. coli expression, synthesized, and cloned into pJexpress401 by DNA2.0. Variants were made with the indicated amino acid substitutions.
  • B. subtilis MEP pathway genes (dxs, dxr, ispD, ispF, and idi) were placed under the control of strong E, coli promoters and cloned into the Hindlll/SacI sites of pCL1920.
  • In vivo isoprene production was measured by injecting flask headspace samples into a Tianmei GC9700 GC equipped with a photoionization detector.
  • the column was a PLOT-Q (Tianmei), the column temperature was 140 C, the injector temperature was 150 C, and the detector temperature was 150 C.
  • the injection volume was 1 mL gas. I soprene standards were used to quantify the signal.
  • Example 4,3 In vivo isoprene production
  • pre-seed cultures single colonies from each strain to be tested were used to inoculate 4 mL LB medium plus appropriate antibiotics and grown overnight at 37 C with shaking. To make seed cultures, for each strain, 300 uL of pre-seed was inoculated into 30 mL MOPS mineral medium (37.9 mM (N i l iLS : , 4,2 mM MgS()
  • 0.5 mL seed culture was diluted into 9.5 mL. fresh MOPS mineral medium plus 5 g/L glucose, 1 g/L yeast extract, appropriate antibiotics, and 1 mM IPTG and incubated at 37 C in an air tight flask with shaking. Isoprene in the headspace was measured after 16 hours.
  • E. globulus isoprene synthase when expressed in E. coli cells, produces far more isoprene than the M. alterniforia or R. pseudoacacia isoprene synthase, or any of the non-isoprene synthases with variants predicted to increase isoprene production.
  • E. coli FM5 strains expressing the indicated terpenoid synthase and heterologous expression of dxs, dxr, ispD, ispF, and idi were grown in minimal medium under aerobic conditions and isoprene production measured (Table 2),
  • E. globulus isoprene synthase The expression and/or solubility of the E. globulus isoprene synthase is far superior to that of M. alterniforia or R. pseuaoacacia isoprene synthases. Total, soluble, and insoluble protein fractions are shown after induction of the indicated plasmids, and the E. globulus isoprene synthase was shown to express at high level in soluble form, unlike the M, alterniforia or R, pseudoacacia isoprene synthases ( Figure 10).
  • E. coli strain FM5-Alper also called strain Q
  • pJexpress404 vector DNA 2.0
  • DNA 2.0 sequence for a histidine- tagged phellandrene synthase (PhS) or a modified version (Mod A) of the His-tagged PhS. Both sequences were behind a T5 promoter.
  • a resulting E. coli transformant containing each PhS sequence was grown in a 250 ml flask of LB overnight atroom temperature (OD ⁇ 1 ). Cultures were induced by adding enough IPTG to bring the culture concentration to 400 ⁇ , Cultures were allowed to grow an additional eight hours until they reached an OD ⁇ 4. Cultures were then concentrated by centrifugation and cells were broken using a combination of freezing, sonication. and lysozyme.
  • Histidine-tagged protein was purified on Ni-agarose (Qiagen) according to
  • DMADP Dimethylallyl diphosphate
  • Assays were carried out in a total volume of 200 LsL of assay buffer (100 niM Hepes pH 7.8, 40 mM KC1, 20 mM MgC12 10% glycerol).
  • DM ADP stock or an equivalent amount of 2 mM NH4HC03 was used to establish different concentrations ofDADP in the assay . There was 20 ,ug protein in each sample.
  • the head- space air was injected into a chemiluminescent isoprene detection system (Fast Isoprene Sensor, Hills Scientific) that had a flowing gas stream, isoprene standards were drawn from a tank of compressed nitrogen with 3.25 ⁇ 0.16 PPM isoprene (Airgas).
  • the isoprene signal from each injection lasted about 15 seconds.
  • Ail of the FlSsignal for 30 seconds centered on the injection peak were summed and 15 seconds of baseline signal before and after the peak were summed and subtracted from the injection signal.
  • the italicized amino acids represent leader sequences that are optionally completely or partially deleted or substituted.
  • the bold amino acids indicate exemplar ⁇ ' sites for modification, singly or in combination. Said modification encompasses substitution, deletion and insertion. Substitutions are indicated using the notation
  • Route 1 making the change S440F, shown in bold and highlighted
  • Route 1 making the change S440Y. shown in bold and highlighted
  • 8709 is shown in bold and highlighted.
  • V33 IF is shown bold and highlighted
  • V444F is shown bold and highlighted
  • V444Y (V331W and V444F), (V331W and V444W), (V331W and V444Y), ( V331Y and V444F), (V331 Y and V444W) or (V331Y and V444Y) (not shown).
  • V331F and V444F are shown bold and highlighted
  • This ⁇ -phellandrene synthase is converted into an isoprene producing enzyme using a combination of one or both of the changes V524F and M672F.
  • V524 and M672 are shown bold, underlined and high-lighted.
  • SEQ ID NO 24 Populus tremuloides isoprene synthase
  • the active site is opened up by making the mutation F338L and F485W (shown bold and underlined), F338L and F485Y, F338W and F485L, F338Y and F485L (not shown)
  • the active site is opened up by making the mutation F338L and F485 W (shown bold and underlined), F338L and F485Y, F338W and F485L, F338Y and F485I, (not shown)
  • the active site is opened up by making the mutation F338L and F485W (shown bold and underlined), F338I, and F485Y, F338W and I 485! ., F338Y and F485L (not shown)
  • the active site is opened up by making the mutation F301L and F448W (shown bold underlined), F301L and F448Y, I 301 W and F448L, F301Y and F448L (not shown)
  • the active site is opened up by making the mutation F343L and F493W (shown bold underlined), F343L and F493Y, F343W and F493L, F343Y and F493L (not shown)
  • Eucalyptus globulus "mts-1 mRNA for nionoterpene synthase" actually an isoprene synthase
  • the active site is opened up by making the mutation F326L and F473W (shown bold and underlined), F326L and F473Y, F326W and F473L, F326Y and F473L (not shown)
  • EKGISELEAR ECVKEEIDTA WKKMNKYMVD RSTFNQSFVR MTYNLARMAH CVYODGDAIG SPDDLSWNRV HSLIIKPISP AA
  • the active site is opened up by making the mutation F326L and F473W (shown bold and underlined), F326L and F473Y, F326W and F473L, F326Y and F473L (not shown)
  • the active site is opened up by making the mutation F281 L and F428W (shown bold underlined), F281L and F428Y, F281W and F428L, F281Y and F428L (not shown)
  • Humul s lupul s (Hops) isoprene synthase
  • F354 and F530 are indicated in bold, underlined and high-lighted
  • Resides that line the active site and can be converted into phenylalanines (F) are show r n bold and highlighted.
  • V 179F is shown in bold and highlighted.
  • N219F is shown in bold and highlighted.
  • V i ' *- ! ⁇ ' and N219F are shown in bold and highlighted.
  • MPQDVDFHIP LPGROSPDHA RAEAEOLAWP RSLGLiRSDA AAERRLRGGY ADLASRFYPH ATGADLDLGV DLMSWFFLFD DLFDGPRGEN PEDTKQLTDQ VAAALDGPLP DTAPPIAHGF ADIWRRTCEG MTPAWCARS RHWRNYFDGY VDSASSRFWN APCDSAAQYL AMRRHTIG ⁇ Q PTVDLAERAG RFEVPHRVFD SAvMSAMLQI AVDVNLLL
  • cgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcg ctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatat atactttagattgatttaaaacttcattttttaattttaaaaggatctaggtgaagatcct ttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcag accccgtagaaaagatcaaaggatcttctttgagatccttttttgcgtttccactga

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Abstract

La présente invention concerne de nouvelles solutions d'industrie chimique et énergétique qui sont renouvelables à la fois écologiquement et économiquement. L'invention concerne l'utilisation d'isoprène synthases variantes conçues pour augmenter la vitesse de production d'isoprène dans des microorganismes génétiquement modifiés ou « biocatalyseurs » servant en tant qu'alternatives viables à des produits chimiques dépendant du pétrole et à de l'énergie dépendante du pétrole.
PCT/US2012/048422 2011-07-26 2012-07-26 Isoprène synthases à efficacité élevée produites par l'ingénierie protéique WO2013016591A1 (fr)

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WO2015076392A1 (fr) 2013-11-22 2015-05-28 味の素株式会社 Isoprène synthase modifiée
WO2017022856A1 (fr) * 2015-08-05 2017-02-09 味の素株式会社 Procédé de production de monomère isoprène
WO2019086466A1 (fr) * 2017-10-31 2019-05-09 Janssen Vaccines & Prevention B.V. Adénovirus et utilisations associées
WO2020102541A1 (fr) * 2018-11-14 2020-05-22 Manus Bio, Inc. Cellules microbiennes et procédés permettant de produire des cannabinoïdes
WO2020234307A1 (fr) * 2019-05-20 2020-11-26 C3 Bio-Technologies Limited Linalol synthases
US11236361B2 (en) 2017-10-31 2022-02-01 Janssen Vaccines & Prevention B.V. Adenovirus and uses thereof
US11459583B2 (en) 2017-10-31 2022-10-04 Janssen Vaccines & Prevention B.V. Adenovirus vectors and uses thereof
US11872281B2 (en) 2017-10-31 2024-01-16 Janssen Vaccines & Prevention B.V. Adenovirus and uses thereof

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015076392A1 (fr) 2013-11-22 2015-05-28 味の素株式会社 Isoprène synthase modifiée
US9890373B2 (en) 2013-11-22 2018-02-13 Ajinomoto Co., Inc. Modified isoprene synthase
WO2017022856A1 (fr) * 2015-08-05 2017-02-09 味の素株式会社 Procédé de production de monomère isoprène
WO2019086466A1 (fr) * 2017-10-31 2019-05-09 Janssen Vaccines & Prevention B.V. Adénovirus et utilisations associées
US11142551B2 (en) 2017-10-31 2021-10-12 Janssen Vaccines & Prevention B.V. Adenovirus and uses thereof
US11236361B2 (en) 2017-10-31 2022-02-01 Janssen Vaccines & Prevention B.V. Adenovirus and uses thereof
US11459583B2 (en) 2017-10-31 2022-10-04 Janssen Vaccines & Prevention B.V. Adenovirus vectors and uses thereof
US11872281B2 (en) 2017-10-31 2024-01-16 Janssen Vaccines & Prevention B.V. Adenovirus and uses thereof
WO2020102541A1 (fr) * 2018-11-14 2020-05-22 Manus Bio, Inc. Cellules microbiennes et procédés permettant de produire des cannabinoïdes
WO2020234307A1 (fr) * 2019-05-20 2020-11-26 C3 Bio-Technologies Limited Linalol synthases
CN114174506A (zh) * 2019-05-20 2022-03-11 C3生物科技有限公司 芳樟醇合酶
CN114174506B (zh) * 2019-05-20 2024-03-15 C3生物科技有限公司 芳樟醇合酶

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